Preliminary Analysis Of Proteomics Of Excretory-Secretory(ES) Products From Trichinella Pseudospiralis

T.pseudospiralis is the zoonotic parasitic nematode that can infect both mammals and birds.People usually infect T.pseudospiralis because they eat raw or undercooked pork and other animals that contain infective muscle larvae.The larvae could not form protective collagen capsule in the host muscle fibers and had no antifreeze.Because it can infect birds,it is distributed globally in nature.Due to the difference between T.pseudospiralis and T.spiralis in the form of no collagen capsule around them,there are significant differences in inflammatory response,phenotype regulation of muscle cells,size of larvae and adults,movement of naked larvae,morphology of stichocyte granules and protein content,especially in the difference of immune specificity,which deserves further study.T.pseudospiralis produces excretes-secretes?ES?products during the parasitic process in the host.ES products are believed to be directly exposed to the host immune system and are the main antigens that induce the host to produce immune response.At present,it has been found that ES products play an important role in penetration of tissues,migration of larvae,regulation of immune cells,slowing down coagulation,digestion,molting and degradation of extracellular matrix.It plays an important role in the interaction between T.pseudospiralis and its host.In this study,flow cytometry was used to detect the changes of immune cell levels in peripheral blood at different infection time points of pig-infected T.pseudospiralis,so as to observe whether different developmental stages of T.pseudospiralis have a strong regulatory effect on host immune response after infection with T.pseudospiralis.Secondly,differential proteomics was used to identify and contrastively analyze the significantly differentially expressed proteins in the ES products of T.pseudospiralis muscle larvae?T4 RUS,T4 AUS and T4 USA?and T.spiralis?T1 ISS534?muscle larvae,and these functional proteins were analyzed.The possible roles of these functional proteins in the interaction between parasites and hosts were discussed in order to find out which ES proteins are involved in the regulation of host immune response,which leads to the stronger ability of T.pseudospiralis to inhibit host inflammation than T.spiralis,and to explain the possible reasons why T.pseudospiralis escapes from host killing.Finally,the specific proteins of adult and newborn larvae of T.pseudospiralis were screened by immunoproteomics as potential early diagnosis antigens or vaccine candidates.In order to observe whether T.pseudospiralis can regulate the host immune system,we used flow cytometry to detect the changes of CD4⁺T cells,CD8⁺T cells,Treg cells,Th17 cells,CD3⁺T cells,CD21⁺B cells,neutrophils and specific cytokine?IL-2 and IL-4?in the peripheral blood of pigs infected with T.pseudospiralis?RUS and AUS?at different infection time points.The results showed that Th2 type cell responses were mainly induced by T.pseudospiralis infection in intestine.B cells may play an important role in cell-mediated response in the stage of chronic infection.Treg cells increased from the 11th day of infection,showing a strong regulatory and inhibitory effect on the host immune response.When T.pseudospiralis invaded the host pigs,the level of inflammatory cells Th17 was slightly higher than that in the blank group at 45 and 60 days,and the rest were lower than that in the blank group.The immune cells in the peripheral blood of pigs infected with T.pseudospiralis RUS and AUS at the same dose?10000 ML?showed the same change trend at different infection time,which caused the same host immune response.There was no significant difference in the trend of other immune cells at different doses?1000 ML and 10000 ML?.We used iTRAQ-MS/MS technology to identify ES products of muscle larvae among three isolates of T.pseudospiralis?RUS,AUS and USA?and T.spiralis?ISS534?.A total of 2591 proteins were identified,of which 535 were credible.A total of 65?146?,72?98?,43?103?and 28?147?differentially expressed proteins were identified in each two groups?T4 RUS:T4 USA,T4 AUS:T4 USA,T4 RUS:T4AUS and T1:T4 USA?,and the identified differentially expressed proteins were analyzed by bioinformatics.In the enrichment analysis of GO and KEGG pathway,we found that the main biological processes involved in differential proteins were carbohydrate metabolic process,small molecule metabolic process,generation of precursor metabolites and energy and protein folding.The main KEGG pathways involved are glycolysis/gluconeogenesis,fructose and mannose metabolism,starch and sucrose metabolism and pentose phosphate pathway;The pathways related to amino acid metabolism include tryptophan metabolism,histidine metabolism,valine,leucine and isoleucine degradation,glycine,serine and threonine metabolism pathway;The pathways related to fat metabolism include pyruvate metabolism,fatty acid metabolism and fatty acid degradation pathway.These biological processes and pathways involved in differential proteins are mainly focused on energy synthesis,metabolism and protein synthesis,which provide support for long-term parasitism of parasites.In addition,we found that the differentially expressed proteins enriched in the lysosome and peroxisome pathways of T4 RUS:T4 USA and T4 AUS:T4 USA groups were all down-regulated.Lysosomes play an important role in signal transduction,cell adaptation,plasma membrane repair,immune response and many other basic cellular processes.In particular,lysosomal mediated degradation is helpful for phagocytes to rapidly clear apoptotic cells and regulate host inflammatory response.However,peroxisome is related to the scavenging of ROS.The expression of all differential proteins involved in lysosomal and peroxisome pathways in T4 USA was significantly higher than that in the other two groups.Searching the String database,we drew the map of the interaction network of different proteins,and found that F23B12.5,Hsp83,calr,T01₁1246,MDH1 and Nom1 are the important nodes in the map of the interaction network,which have close interaction with multiple proteins and function in cooperation.Functional classification of the identified differential proteins revealed some proteins involved in antioxidant,anti-stress,host immune regulation and signal transduction.For example,antioxidant proteins include protein disulfide isomerase?PPI?and peroxiredoxin,anti-stress proteins include heat shock proteins HSP70,Hsp83,HSP beta-1 and alpha crystal B chain?small HSP?,DNase?involved in host immune regulation,and proteins involved in Ca²⁺signal transduction and stability,including calmodulin,calreticulin and calsyntenin-1.Compared with the expression of these functional differential proteins,we found that the expression of T4 USA was significantly higher than that of the other three groups.We found RWD domain-containing protein 2B?97%query cover?which was highly homologous with 53kDa glycoprotein,which can stimulate the host strong Th2 type reaction,inhibit the Th1 reaction,and increase the Th2 cytokines IL-4 and IL-13.Whether the protein found in ES products of T.pseudospiralis performs similar protein functions needs to be further verified.Finally,we used 2DE-western blot combined with MALDI-TOF/TOF-MS/MS to identify ES products of adult worm and newborn larvae of T.pseudospiralis,and identified about 24 matching protein spots through PMF of peptide fingerprint,and identified 12 different proteins.GO annotation was used to analyze the main molecular functions of these proteins involved in DNase II activity and serine type endonuclease activity.We found that DNase II and serine protease related to immune regulation,PCHTP protein which can combine with metal ions,venom allergen 5protein is conducive to adult worm escape to host and enolase is a virulence factor of invading host.Serine protease in ES product of microfilariae of the maleosma confirmed that it can inhibit the aggregation of inflammatory cells and the release of inflammatory factors,so as to mediate the immunosuppression of the host.In this experiment,several serine proteases found in ES products of adult and newborn larvae may also participate in the immunosuppression of the host,and we will continue to verify it in the following studies.These proteins can be used as candidate proteins for early diagnosis or vaccine.In conclusion,ES products of T.pseudospiralis at different developmental stages?ML,Ad and NBL?were identified and their functional proteins were analyzed.It is important to understand the immunosuppressive ability of T.pseudospiralis to host,the interaction between parasite and host,and the screening of early potential diagnostic proteins.